Next-generation sequencing for JAK2 mutation testing: advantages and pitfalls.

The JAK2V617F mutation is part of the major criteria for diagnosis of myeloproliferative neoplasms (MPN). Allele-specific quantitative PCR (qPCR) is the most prevalent method used in laboratories but with the advent of next-generation sequencing (NGS) techniques, we felt necessary to evaluate this approach for JAK2 mutations testing. Among DNA samples from 427 patients analyzed by qPCR and NGS, we found an excellent concordance between both methods when allelic burden was superior to 2% (the detection limit of our NGS assay). Only one sample among 298 was found negative by NGS while allelic burden by qPCR was 3%. Because NGS detection limit is higher, sensitivity was lower as exemplified by 21 samples found negative whereas qPCR measured allelic burdens between 0.1 and 1%. Importantly, quantitative data of samples found positive by both techniques were highly correlated (R2 = 0.9477). We also evaluated 40 samples tested for JAK2 exon 12 mutations by HRM. The concordance with NGS was of 100%. Using NGS, the full coding region of JAK2 was analyzed leading to identification of several variants outside of exon 12 and 14 which were previously described or not. Interestingly, we found one somatic mutation (c.1034A>T p.H345L) which induced constitutive activation of the JAK/STAT pathway leading to an increased proliferation of BaF/3 cells with low-dose EPO. This study showed that NGS is a robust method highly correlated to qPCR, although less sensitive, but providing the opportunity to identify other JAK2 variants with potential impact on disease initiation or evolution.

A nationwide survey of the use of plerixafor in patients with lymphoid malignancies who mobilize poorly demonstrates the predominant use of the "on-demand" scheme of administration at French autologous hematopoietic stem cell transplant programs.

BACKGROUND: High-dose chemotherapy supported with autologous stem cell transplantation is a standard therapeutic option for a subset of patients with lymphoid malignancies. Cell procurement is nowadays done almost exclusively through cytapheresis, after mobilization of hematopoietic stem and progenitor cells (HSPCs) from the marrow to peripheral blood (PB). The egress of HSPCs out of hematopoietic niches occurs in various physiologic or nonhomeostatic situations; pharmacologic approaches include the administration of acutely myelosuppressive agents or hematopoietic growth factors such as recombinant human granulocyte-colony-stimulating factor (rHuG-CSF). The introduction of plerixafor, a first-of-its-class molecule that reversibly inhibits the interaction between the chemokine CXCL-12 (also known as SDF-1) and its receptor CXCR-4, has offered new opportunities for the so-called "poor mobilizers" who achieve insufficient mobilization and/or collection with conventional approaches. STUDY DESIGN AND METHODS: Because of the lack of consensus on a definition for poor mobilizers and the relatively high cost of plerixafor, French competent authorities have mandated a postmarketing survey on its use in routine practice. RESULTS AND CONCLUSION: We report here the results of this nationwide survey that confirms the clinical efficacy of plerixafor, even in the subset of patients who barely increased PB CD34+ cell count in response to rHuG-CSF-containing mobilization regimen. Furthermore, analysis of this registry showed that despite heterogeneity in medical practices, the early-"on-demand" or "preemptive"-introduction of plerixafor was widely used and did not result in an excess of prescriptions, beyond its expected use at the time when marketing authorization was granted.

A specific time course for mobilization of peripheral blood CD34+ cells after plerixafor injection in very poor mobilizer patients: impact on the timing of the apheresis procedure.

BACKGROUND: This report describes the specific kinetics of the peripheral blood (PB) CD34+ cell concentration in a selected group of very poor stem cell mobilizer patients treated with granulocyte-colony-stimulating factor (G-CSF) and plerixafor and determines the kinetics' impact on apheresis. STUDY DESIGN AND METHODS: All patients had previously experienced at least two failures of mobilization (without use of plerixafor). The present salvage therapy consisted in the administration of 10 µg/kg/day G-CSF for 5 days added to a dose of plerixafor administered at between 5 a.m. and 6 a.m. on Day 5. The PB CD34+ cell counts were tested every 3 hours thereafter. Apheresis was initiated as soon as the PB CD34+ cell count reached 10 × 10(6) /L. RESULTS: A PB CD34+ cell count higher than 10 × 10(6) /L was observed as soon as 3 hours after plerixafor administration in 10 of the 11 patients who reached this threshold at some point in the monitoring process. Interestingly, all patients presented an early decrease in the PB CD34+ cell count 8 to 12 hours after plerixafor administration (below 10 × 10(6) /L for seven patients). CONCLUSION: Had such patients been tested for PB CD34+ cell mobilization according to conventional criteria (i.e., 11 hr after plerixafor administration), apheresis would not have been performed at the optimal timing. For very poor stem cell mobilizer patients, early monitoring of PB CD34+ cell count may be required for the optimal initiation of apheresis.

Impact of rituximab on stem cell mobilization following ACVBP regimen in poor-risk patients with diffuse large B-cell lymphoma: results from a large cohort of patients.

BACKGROUND: The ACVBP regimen is an efficient induction regimen for poor-risk patients with diffuse large B-cell lymphoma (DLBCL) before consolidative autologous stem cell transplantation. Adjunction of the monoclonal anti-CD20 antibody rituximab (R-ACVBP) was recently found to be superior to ACVBP alone. This study assessed the impact of rituximab on stem cell mobilization in two similar consecutive groups of patients treated with ACVBP in two prospective, controlled trials. STUDY DESIGN AND METHODS: The first trial (LNH-98B-3) involved 137 patients treated with ACVBP alone. In the second trial (LNH-03-3B), 91 patients received an R-ACVBP regimen. Stem cell mobilization was performed after a course of (R)-ACVBP. RESULTS: The median peak numbers of blood CD34+ cell counts recorded before the first apheresis procedure in the ACVBP and R-ACVBP groups were 69×10(6) and 63×10(6) /L, respectively (p=0.55). The median numbers of CD34+ cells collected were 7.1×10(6) and 6.0×10(6) CD34+ cells/kg for the ACVBP and R-ACVBP groups, respectively (p=0.13). The median number of apheresis procedures required for gathering the minimum amount of CD34+ cells (2×10(6) /kg) was the same in the two groups. CONCLUSION: When compared with ACVBP alone, adjunction of rituximab does not impair stem cell mobilization.

Therapy-related classical Hodgkin lymphoma after a primary haematological malignancy: a report on 13 cases.

The risk of developing Hodgkin lymphoma (HL) is increased in immunodeficiencies or during the treatment of some autoimmune diseases. The development of new therapeutic agents has highlighted the risk of unusual lymphoid proliferations, particularly classical HL (cHL). We report the clinicopathological findings of 13 cHL arising in patients treated for a primary haematological malignancy. Eight patients had received an immunomodulator, protein tyrosine-kinase inhibitor or monoclonal antibody, which may have contributed to the cHL development. Most patients had disseminated disease with poor prognostic factors at cHL diagnosis. Despite the initial presentation, good outcomes were achieved with standard cHL chemotherapy.

Long-term outcome of imatinib 400 mg compared to imatinib 600 mg or imatinib 400 mg daily in combination with cytarabine or pegylated interferon alpha 2a for chronic myeloid leukaemia: results from the French SPIRIT phase III randomised trial.

The STI571 prospective randomised trial (SPIRIT) French trial is a four-arm study comparing imatinib (IM) 400 mg versus IM 600 mg, IM 400 mg + cytarabine (AraC), and IM 400 mg + pegylated interferon alpha2a (PegIFN-α2a) for the front-line treatment of chronic-phase chronic myeloid leukaemia (CML). Long-term analyses included overall and progression-free survival, molecular responses to treatment, and severe adverse events. Starting in 2003, the trial included 787 evaluable patients. The median overall follow-up of the patients was 13.5 years (range 3 months to 16.7 years). Based on intention-to-treat analyses, at 15 years, overall and progression-free survival were similar across arms: 85%, 83%, 80%, and 82% and 84%, 87%, 79%, and 79% for the IM 400 mg (N = 223), IM 600 mg (N = 171), IM 400 mg + AraC (N = 172), and IM 400 mg + PegIFN-α2a (N = 221) arms, respectively. The rate of major molecular response at 12 months and deep molecular response (MR4) over time were significantly higher with the combination IM 400 mg + PegIFN-α2a than with IM 400 mg: p = 0.0001 and p = 0.0035, respectively. Progression to advanced phases and secondary malignancies were the most frequent causes of death. Toxicity was the main reason for stopping AraC or PegIFN-α2a treatment.

Successful peripheral blood stem cell harvesting with granulocyte colony-stimulating factor alone after previous mobilization failure.

A total of 138 patients whose stem cell mobilization failed following chemotherapy and granulocyte colony--stimulating factor (G-CSF) at a dose of 5 microg/kg/d were given a higher dose of G-CSF (10 microg/kg/d) for 5 days after a 7-day resting period. Stem cell mobilization was successful in 90 patients, who yielded a median of 3.5x10(6) CD34(+) cells/kg, partially successful in 17 patients (1-2.4x10(6) CD34+ cells/kg) and failed in the remaining 31 patients.

Rescue therapy combining intermediate-dose cytarabine with amsacrine and etoposide in relapsed adult acute lymphoblastic leukemia.

In all, 625 patients with acute lymphoblastic leukemia (ALL) entered the Leucémie Aiguë Lymphoblastique de l'Adulte-94 trial from June 1994 to June 1999, and received a 4-week induction therapy followed either by chemotherapy alone or stem cell transplantation (SCT). In a clinical phase II study, 40 patients with standard- or high-risk ALL - except Philadelphia chromosome-positive ALL -, relapsing at least 3 months after the beginning of therapy and who did not receive any SCT, received a rescue protocol combining amsacrine 120 mg/m(2)/day, days 1-3, cytarabine 1 g/m(2)/12 h, days 1-5, and etoposide 100 mg/m(2)/day, days 1-5. All relapses occurred 'on therapy'. In all, 16 patients (40%) achieved a second complete remission. The median time to neutrophil recovery >0.5 x 10(9)/l was 27 days. The median time to platelet recovery >50 x 10(9)/l was 28 days. Extra-hematologic toxicity was mild (only one toxic death from severe infection). The median overall survival was 5.4 months. The median disease-free survival (DFS) was 3.2 months with a 3-year DFS of 12%. Unfavorable prognostic factors for complete remission achievement were: high-risk ALL at diagnosis (P=0.03), and white blood cell count at relapse >or=30 x 10(9)/l (P=0.02). No relationship was found between survival and any characteristics of the disease. Four patients underwent allogeneic SCT (two phenoidentical and two genoidentical) and three patients received autologous SCT. This treatment combining amsacrine, cytarabine, and etoposide was therefore effective and well tolerated in 'on-therapy'-relapsed ALL. However, the median DFS was short requiring the rapid completion of effective intensive postremission therapy.

Long term hematologic recovery after autologous stem cell transplantation in lymphoma patients: impact of the number of prefreeze and post-thaw CD34+ cells.

BACKGROUND: Autologous Stem Cell Transplantation (ASCT) with Peripheral Blood Stem Cells is widely used as consolidation in lymphoma patients. The rapidity and stability of cell engraftment correlate with the number of CD34+ cells in the autograft. However, whether CD34+ cells should be quantified before or after cryopreservation remains unclear. PATIENTS AND METHODS: Of 173 consecutive patients who underwent ASCT in our department from Nov 1, 1995 to Nov 1, 2000, 133 (78 %) were alive without relapse at one year. We report here the results for 106 patients whose hematologic data were available. RESULTS: At one year, the hemoglobin was normal in 47% of the patients, the leukocytes, in 77% and the platelets, in 60%. Only 33% had a normal blood count. We observed a significant correlation between prefreeze and post-thaw CD34+ cell numbers (r = 0.77). However, multivariate analysis using the Cox model with smoothing splines to assess the best cut-off point for these numbers demonstrated that the only independent predictive factor for a normal blood count after one year was a prefreeze number of CD34+ cells above 5.10(6)/kg. CONCLUSION: An optimal long-term hematologic recovery after ASCT required a number of prefreeze CD34+ cells of at least 5.10(6)/kg.

Randomized comparison of double induction and timed-sequential induction to a "3 + 7" induction in adults with AML: long-term analysis of the Acute Leukemia French Association (ALFA) 9000 study.

Between 1990 and 1996, we conducted a randomized trial in adults with newly diagnosed acute myeloid leukemia (AML) in order to compare relapse-free interval (RFI) after double induction (arm B), timed-sequential induction (arm C), or control "3 + 7" induction (arm A). Patients achieving complete remission (CR) after induction +/- salvage received the same consolidation chemotherapy, which included a dosage stratification according to patient's age (younger or older than 50 years). This long-term analysis was performed in 592 patients (arm A/B/C, 197/198/197 patients). Overall CR rate was 76% without differences between the 3 arms, even if a salvage course was less frequently needed in arm B. Treatment-related mortality, either during the induction or the postremission phase, was not significantly higher in arms B and C than in arm A. Among the 449 CR patients, 250 relapsed (arm A/B/C, 90/87/73 patients) without significant differences in RFI in arms B and C versus arm A (P = .39 and .15, by the Gray test). However, when analyzing the 345 patients younger than 50, RFI was significantly improved in younger patients receiving timed-sequential induction (P = .038 by the Gray test), while not in those receiving double induction. Event-free survival and overall survival were similar in the 3 randomization arms.